The effect of oral metformin on insulin sensitivity in insulin-resistant ponies

Metformin may be an effective therapeutic option for insulin-resistant (I-R) horses/ponies because, in humans, it reportedly enhances insulin sensitivity (SI) of peripheral tissues without stimulating insulin secretion. To determine the effect of metformin on insulin and glucose dynamics in I-R ponies, six ponies were studied in a cross-over design by Minimal Model analysis of a frequently-sampled intravenous glucose tolerance test (FSIGT). Metformin was administered at 15. mg/kg bodyweight (BW), orally, twice-daily, for 21. days to the metformin-treated group. The control group received a placebo. A FSIGT was conducted before and after treatment. The Minimal Model of glucose and insulin dynamics rendered indices describing SI, glucose effectiveness (Sg), acute insulin response to glucose (AIRg) and the disposition index (DI). The body condition score (BCS), BW and cresty neck score (CNS) were also assessed. There was no significant change in SI, Sg, AIRg, DI, BW, BCS or CNS in response to metformin, or over time in the control group. There were no measurable benefits of metformin on SI, consistent with recent work showing that the bioavailability of metformin in horses is poor, and chronic dosing may not achieve therapeutic blood concentrations. Alternatively, metformin may only be effective in obese ponies losing weight or with hyperglycaemia.

The developmental and acute phases of insulin-induced laminitis involve minimal metalloproteinase activity

Metalloproteinases have been implicated in the pathogenesis of equine laminitis and other inflammatory conditions, through their role in the degradation and remodelling of the extracellular matrix environment. Matrix metalloproteinases (MMPs) and their inhibitors are present in normal equine lamellae, with increased secretion and activation of some metalloproteinases reported in horses with laminitis associated with systemic inflammation. It is unknown whether these enzymes are involved in insulin-induced laminitis, which occurs without overt systemic inflammation. In this study, gene expression of MMP-2, MMP-9, MT1-MMP, ADAMTS-4 and TIMP-3 was determined in the lamellar tissue of normal control horses (n = 4) and horses that developed laminitis after 48 h of induced hyperinsulinaemia (n = 4), using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Protein concentrations of MMP-2 and MMP-9 were also examined using gelatin zymography in horses subject to prolonged hyperinsulinaemia for 6 h (n = 4), 12 h (n = 4), 24 h (n = 4) and 48 h (n = 4), and in normal control horses (n = 4). The only change in gene expression observed was an upregulation of MMP-9 (p < 0.05) in horses that developed insulin-induced laminitis (48 h). Zymographical analysis showed an increase (p < 0.05) in pro MMP-9 during the acute phase of laminitis (48 h), whereas pro MMP-2 was present in similar concentration in the tissue of all horses. Thus, MMP-2, MT1-MMP, TIMP-3 and ADAMTS-4 do not appear to play a significant role in the pathogenesis of insulin-induced laminitis. The increased expression of MMP-9 may be associated with the infiltration of inflammatory leukocytes, or may be a direct result of hyperinsulinaemia. The exact role of MMP-9 in basement membrane degradation in laminitis is uncertain as it appears to be present largely in the inactive form.

Acepromazine pharmacokinetics: A forensic perspective

Acepromazine (ACP) is a useful therapeutic drug, but is a prohibited substance in competition horses. The illicit use of ACP is difficult to detect due to its rapid metabolism, so this study investigated the ACP metabolite 2-(1-hydroxyethyl)promazine sulphoxide (HEPS) as a potential forensic marker. Acepromazine maleate, equivalent to 30 mg of ACP, was given IV to 12 racing-bred geldings. Blood and urine were collected for 7 days post-administration and analysed for ACP and HEPS by liquid chromatography–mass spectrometry (LC–MS). Acepromazine was quantifiable in plasma for up to 3 h with little reaching the urine unmodified. Similar to previous studies, there was wide variation in the distribution and metabolism of ACP. The metabolite HEPS was quantifiable for up to 24 h in plasma and 144 h in urine. The metabolism of ACP to HEPS was fast and erratic, so the early phase of the HEPS emergence could not be modelled directly, but was assumed to be similar to the rate of disappearance of ACP. However, the relationship between peak plasma HEPS and the y-intercept of the kinetic model was strong (P = 0.001, r2 = 0.72), allowing accurate determination of the formation pharmacokinetics of HEPS. Due to its rapid metabolism, testing of forensic samples for the parent drug is redundant with IV administration. The relatively long half-life of HEPS and its stable behaviour beyond the initial phase make it a valuable indicator of ACP use, and by determining the urine-to-plasma concentration ratios for HEPS, the approximate dose of ACP administration may be estimated.

Histological and morphometric lesions in the pre-clinical, developmental phase of insulin-induced laminitis in Standardbred horses

Lamellar pathology in experimentally-induced equine laminitis associated with euglycaemic hyperinsulinaemia is substantial by the acute, clinical phase (∼48 h post-induction). However, lamellar pathology of the developmental, pre-clinical phase requires evaluation. The aim of this study was to analyse lamellar lesions both qualitatively and quantitatively, 6, 12 and 24 h after the commencement of hyperinsulinaemia. Histological and histomorphometrical analyses of lamellar pathology at each time-point included assessment of lamellar length and width, epidermal cell proliferation and death, basement membrane (BM) pathology and leucocyte infiltration. Archived lamellar tissue from control horses and those with acute, insulin-induced laminitis (48 h) was also assessed for cellular proliferative activity by counting the number of cells showing positive nuclear immuno labelling for TPX2. Decreased secondary epidermal lamellar (SEL) width and increased histomorphological evidence of SEL epidermal basal (and supra-basal) cell death occurred early in disease progression (6 h). Increased cellular proliferation in SELs, infiltration of the dermis with small numbers of leucocytes and BM damage occurred later (24 and 48 h). Some lesions, such as narrowing of the SELs, were progressive over this time period (6–48 h). Cellular pathology preceded leucocyte infiltration and BM pathology, indicating that the latter changes may be secondary or downstream events in hyperinsulinaemic laminitis.

Uniform Civil Procedure Rules r150 : orders relating to "property" – whether sperm extracted and stored may be characterised as “property”

In Bazley v Wesley Monash IVF Pty Ltd [2010] QSC 118 an order was made under r 250 of the Uniform Civil Procedure Rules 1999 (Qld) (“UCPR”) requiring the respondent to continue to hold and maintain straws of semen belonging to the applicant’s deceased husband. The decision includes a useful analysis of the development of the common law regarding property rights in human bodies and body parts.

A potential role for lamellar insulin-like growth factor-1 receptor in the pathogenesis of hyperinsulinaemic laminitis

The reason why a sustained high concentration of insulin induces laminitis in horses remains unclear. Cell proliferation occurs in the lamellae during insulin-induced laminitis and in other species high concentrations of insulin can activate receptors for the powerful cell mitogen, insulin-like growth factor (IGF)-1. The first aim of this study was to determine if IGF-1 receptors (IGF-1R) are activated in the hoof during insulin-induced laminitis. Gene expression for IGF-1R and the insulin receptor (InsR) was measured using qRT-PCR, in lamellar tissue from control horses and from horses undergoing a prolonged euglycaemic, hyperinsulinaemic clamp (p-EHC), during the mid-developmental (24 h) and acute (46 h) phases of insulin-induced laminitis. Gene expression for both receptors was decreased 13–32-fold (P < 0.05) at both time-points in the insulin-treated horses. A second aim was to determine if the down-regulation of the receptor genes could be accounted for by an increase in circulating IGF-1. Serum IGF-1 was measured at 0, 10, 25 and 46 h post-treatment in horses given a p-EHC for approximately 46 h, and in matched controls administered a balanced, electrolyte solution. There was no increase in serum IGF-1 concentrations during the p-EHC, consistent with down-regulation of both receptors by insulin. Stimulation of the IGF-1R by insulin may lead to inappropriate lamellar epidermal cell proliferation and lamellar weakening, a potential mechanism for hyperinsulinaemic laminitis. Targeting this receptor may provide insights into the pathogenesis or identify a novel therapy for hyperinsulinaemic laminitis.

The feral horse foot. Part B : radiographic, gross visual and histopathological parameters of foot health in 100 Australian feral horses

Background It has been proposed that the feral horse foot is a benchmark model for foot health in horses. However, the foot health of feral horses has not been formally investigated. Objectives To investigate the foot health of Australian feral horses and determine if foot health is affected by environmental factors, such as substrate properties and distance travelled. Methods Twenty adult feral horses from five populations (n = 100) were investigated. Populations were selected on the basis of substrate hardness and the amount of travel typical for the population. Feet were radiographed and photographed, and digital images were surveyed by two experienced assessors blinded to each other's assessment and to the population origin. Lamellar samples from 15 feet from three populations were investigated histologically for evidence of laminitis. Results There was a total of 377 gross foot abnormalities identified in 100 left forefeet. There were no abnormalities detected in three of the feet surveyed. Each population had a comparable prevalence of foot abnormalities, although the type and severity of abnormality varied among populations. Of the three populations surveyed by histopathology, the prevalence of chronic laminitis ranged between 40% and 93%. Conclusions Foot health appeared to be affected by the environment inhabited by the horses. The observed chronic laminitis may be attributable to either nutritional or traumatic causes. Given the overwhelming evidence of suboptimal foot health, it may not be appropriate for the feral horse foot to be the benchmark model for equine foot health.

Adaption of horses to a novel dynamic feeding system : movement and behavioural responses

Reasons for performing study: Many domestic horses and ponies are sedentary and obese due to confinement to small paddocks and stables and a diet of infrequent, high-energy rations. Severe health consequences can be associated with this altered lifestyle. Objectives: The aims of this study were to investigate the ability of horses to learn to use a dynamic feeder system and determine the movement and behavioural responses of horses to the novel system. Methods: A dynamic feed station was developed to encourage horses to exercise in order to access ad libitum hay. Five pairs of horses (n = 10) were studied using a randomised crossover design with each pair studied in a control paddock containing a standard hay feeder and an experimental paddock containing the novel hay feeder. Horse movement was monitored by a global positioning system (GPS) and horses observed and their ability to learn to use the system and the behavioural responses to its use assessed. Results: With initial human intervention all horses used the novel feeder within 1 h. Some aggressive behaviour was observed between horses not well matched in dominance behaviour. The median distance walked by the horses was less (P = 0.002) during a 4 h period (117 [57–185] m) in the control paddock than in the experimental paddock (630 [509–719] m). Conclusions: The use of an automated feeding system promotes increased activity levels in horses housed in small paddocks, compared with a stationary feeder.

Histopathological examination of chronic laminitis in Kaimanawa feral horses of New Zealand

AIMS: To investigate the prevalence, histopathological and histomorphometric presentation of chronic laminitis in a population of Kaimanawa feral horses. METHODS: Following the capture and euthanasia of feral horses from the Kaimanawa Ranges of New Zealand, the left forefoot of 28 stallions and 28 mares aged between 6 and 12 years were removed and processed for histology. Sections of lamellar samples from each horse were examined using light microscopy. The presence of laminitis was assessed and the histopathological lesions were described. Horses were grouped by histological diagnosis into laminitic and non-laminitic groups and histomorphometric analysis was conducted and compared between groups. The parameters examined were total length of primary epidermal lamellae (PEL), keratinised length of PEL, and the length of secondary epidermal lamellae (SEL) at the abaxial end and axial end of each PEL. RESULTS: Of the horses examined, 25 (45%) were diagnosed with chronic laminitis. The most prevalent histopathological features were the presence of excessive cap horn, and multi-branched and attenuated SEL. Histomorphometric assessment of the lamellar architecture revealed no difference in morphometric measurements between the normal and laminitic groups for any parameter measured (p>0.05). CONCLUSIONS: The current study found a high prevalence of laminitis in feral Kaimanawa horses. The reason for this in the Kaimanawa population is not known. Histomorphometric analysis may not be a good indicator of chronic laminitis in feral horses. CLINICAL RELEVANCE: It is an important finding that the feral horse lifestyle in the environment of the Kaimanawa Ranges in New Zealand offers no protection against foot disease. The finding suggests that horses are vulnerable to laminitis whether in domestic care or in a feral habitat.

Insulin and the insulin-like growth factor system : a novel theory for hyperinsulinemic laminitis pathogenesis

Research into hyperinsulinemic laminitis has progressed significantly in recent years with the use of the prolonged-euglycemic, hyperinsulinemic clamp (p-EHC). Previous investigations of laminitis pathophysiology have focused on digital vascular dysfunction, inflammation, altered glucose metabolism within the lamellae, and lamellar basement membrane breakdown by metalloproteinases. The etiopathogenesis of laminitis occurring in association with hyperinsulinemia is yet to be fully characterized, but it may not involve these mechanisms. Insulin stimulates cellular proliferation and can also affect other body systems, such as the insulin-like growth factor (IGF) system. Insulin-like growth factor-1 (IGF-1) is structurally homologous to insulin and, like insulin, binds with strong affinity to a specific tyrosine kinase receptor on the cell surface to produce its effects, which include promoting cell proliferation. Receptors for IGF-1 (IGF-1R) are present in the lamellar epidermis. An alternative theory for the pathogenesis of hyperinsulinemic laminitis is that uncontrolled cell proliferation, mediated through both the insulin receptor (InsR) and IGF-1R, leads to lengthening, weakening, and failure of the lamellae. An analysis of the proliferative activity of lamellar epidermal cells during the developmental and acute phases of hyperinsulinemic laminitis, and lamellar gene expression of the InsR and IGF-1R was undertaken.

Sole depth and weight-bearing characteristics of the palmar surface of the feet of feral horses and domestic Thoroughbreds

Objective: To determine solar load-bearing structures in the feet of feral horses and investigate morphological characteristics of the sole in feral horses and domestic Thoroughbreds. Sample: Forelimbs from cadavers of 70 feral horses and 20 domestic Thoroughbreds in Australia. Procedures: Left forefeet were obtained from 3 feral horse populations from habitats of soft substrate (SS [n = 10 horses]), hard substrate (HS [10]), and a combination of SS and HS (10) and loaded in vitro. Pressure distribution was measured with a pressure plate. Sole depth was measured at 12 points across the solar plane in feet obtained from feral horses from SS (n = 20 horses) and HS (20) habitats and domestic Thoroughbreds (20). Results: Feet of feral horses from HS habitats loaded the periphery of the sole and hoof wall on a flat surface. Feral horses from HS or SS habitats had greater mean sole depth than did domestic Thoroughbreds. Sole depth was greatest peripherally and was correlated with the loading pattern. Conclusions and Clinical Relevance: The peripheral aspect of the sole in the feet of feral horses had a load-bearing function. Because of the robust nature of the tissue architecture, the hoof capsule of feral horses may be less flexible than that of typical domestic horses. The application of narrow-web horseshoes may not take full advantage of the load-bearing and force-dissipating properties of the peripheral aspect of the sole. Further studies are required to understand the effects of biomechanical stimulation on the adaptive responses of equine feet.

Distances travelled by feral horses in ‘outback’ Australia

Reasons for performing study: The distance travelled by Australian feral horses in an unrestricted environment has not previously been determined. It is important to investigate horse movement in wilderness environments to establish baseline data against which the movement of domestically managed horses and wild equids can be compared. Objectives: To determine the travel dynamics of 2 groups of feral horses in unrestricted but different wilderness environments. Methods: Twelve feral horses living in 2 wilderness environments (2000 vs. 20,000 km2) in outback Australia were tracked for 6.5 consecutive days using custom designed, collar mounted global positioning systems (GPS). Collars were attached after darting and immobilising the horses. The collars were recovered after a minimum of 6.5 days by re-darting the horses. Average daily distance travelled was calculated. Range use and watering patterns of horses were analysed by viewing GPS tracks overlaid on satellite photographs of the study area. Results: Average distance travelled was 15.9 ± 1.9 km/day (range 8.1–28.3 km/day). Horses were recorded up to 55 km from their watering points and some horses walked for 12 h to water from feeding grounds. Mean watering frequency was 2.67 days (range 1–4 days). Central Australian horses watered less frequently and showed a different range use compared to horses from central Queensland. Central Australian horses walked for long distances in direct lines to patchy food sources whereas central Queensland horses were able to graze close to water sources and moved in a more or less circular pattern around the central water source. Conclusions: The distances travelled by feral horses were far greater than those previously observed for managed domestic horses and other species of equid. Feral horses are able to travel long distances and withstand long periods without water, allowing them to survive in semi-arid conditions.

Load bearing improves the limits of agreement for repeated in vivo measures of the speed of sound in human Achilles tendon

Axial acoustic wave propagation has been widely used in evaluating the mechanical properties of human bone in vivo. However, application of this technique to monitor soft tissues, such as tendon, has received comparatively little scientific attention. Laboratory-based research has established that axial acoustic wave transmission is not only related to the physical properties of equine tendon but is also proportional to tensile load to which it is exposed (Miles et al., 1996; Pourcelot et al., 2005). The reproducibility of the technique for in vivo measurements in human tendon, however, has not been established. The aim of this study was to evaluate the limits of agreement for repeated measures of the speed of sound (SoS) in human Achilles tendon in vivo. Methods: A custom built ultrasound device, consisting of an A-mode 1MHz emitter and two regularly spaced receivers, was used to measure the SoS in the mid-portion of the Achilles tendon in ten healthy males and ten females (mean age: 33.8 years, range 23-56 yrs; height: 1.73±0.08 m; weight: 68.4±15.3 kg). The emitter and receivers were held at fixed positions by a polyethylene frame and maintained in close contact with the skin overlying the tendon by means of elasticated straps. Repeated SoS measurements were taken with the subject prone (non-weightbearing and relaxed Achilles tendon) and during quiet bipedal and unipedal stance. In each instance, the device was detached and repositioned prior to measurement. Results: Limits of agreement for repeated SoS measures during non-weightbearing and bipedal and unipedal stance were ±53, ±28 and ±21 m/s, respectively. The average SoS in the non-weightbearing Achilles tendon was 1804±198 m/s. There was a significant increase in the average SoS during bilateral (2122±135 m/s) (P < 0.05) and unilateral (2221±79 m/s) stance (P < 0.05). Conclusions: Repeated SoS measures in human Achilles tendon were more reliable during stance than under non-weightbearing conditions. These findings are consistent with previous research in equine tendon in which lower variability in SoS was observed with increasing tensile load (Crevier-Denoix et al, 2009). Since the limits of agreement for Achilles tendon SoS are nearly 5% of the changes previously observed during walking and therapeutic heel raise exercises, acoustic wave transmission provides a promising new non-invasive method for determining tendon properties during sports and rehabilitation related activities.

An objective measure of reactive behaviour in horses

Several tests have been devised in an attempt to detect behaviour modification due to training, supplements or diet in horses. These tests rely on subjective observations in combination with physiological measures, such as heart rate (HR) and plasma cortisol concentrations, but these measures do not definitively identify behavioural changes. The aim of the present studies was to develop an objective and relevant measure of horse reactivity. In Study 1, HR responses to auditory stimuli, delivered over 6 days, designed to safely startle six geldings confined to individual stalls was studied to determine if peak HR, unconfounded by physical exertion, was a reliable measure of reactivity. Both mean (±SEM) resting HR (39.5 ± 1.9 bpm) and peak HR (82 ± 5.5 bpm) in response to being startled in all horses were found to be consistent over the 6 days. In Study 2, HR, plasma cortisol concentrations and speed of departure from an enclosure (reaction speed (RS)) in response to a single stimulus of six mares were measured when presented daily over 6 days. Peak HR response (133 ± 4 bpm) was consistent over days for all horses, but RS increased (3.02 ± 0.72 m/s on Day 1 increasing to 4.45 ± 0.53 m/s on Day 6; P = 0.005). There was no effect on plasma cortisol, so this variable was not studied further. In Study 3, using the six geldings from Study 1, the RS test was refined and a different startle stimulus was used each day. Again, there was no change in peak HR (97.2 ± 5.8 bpm) or RS (2.9 ± 0.2 m/s on Day 1 versus 3.0 ± 0.7 m/s on Day 6) over time. In the final study, mild sedation using acepromazine maleate (0.04 mg/kg BW i.v.) decreased peak HR in response to a startle stimulus when the horses (n = 8) were confined to a stall (P = 0.006), but not in an outdoor environment when the RS test was performed. However, RS was reduced by the mild sedation (P = 0.02). In conclusion, RS may be used as a practical and objective test to measure both reactivity and changes in reactivity in horses.

Toll-like receptor and pro-inflammatory cytokine expression during prolonged hyperinsulinaemia in horses : implications for laminitis

Equine laminitis, a disease of the lamellar structure of the horse’s hoof, can be incited by numerous factors that include inflammatory and metabolic aetiologies. However, the role of inflammation in hyperinsulinaemic laminitis has not been adequately defined. Tolllike receptor (TLR) activation results in up-regulation of inflammatory pathways and the release of pro-inflammatory cytokines, including interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-�), and may be a pathogenic factor in laminitis. The aim of this study was to determine whether TLR4 expression and subsequent pro-inflammatory cytokine production is increased in lamellae and skeletal muscle during equine hyperinsulinaemia. Standardbred horses were treated with either a prolonged, euglycaemic hyperinsulinaemic clamp (p-EHC) or a prolonged, glucose infusion (p-GI), which induced marked and moderate hyperinsulinaemia, respectively. Age-matched control horses were treated simultaneously with a balanced electrolyte solution. Treated horses developed clinical (p-EHC) or subclinical (p-GI) laminitis, whereas controls did not. Skeletal muscle and lamellar protein extracts were analysed by Western blotting for TLR4, IL-6, TNF-� and suppressor of cytokine signalling 3 (SOCS3) expression. Lamellar protein expression of TLR4 and TNF-�, but not IL-6, was increased by the p-EHC, compared to control horses. A significant positive correlation was found between lamellar TLR4 and SOCS3. Skeletal muscle protein expression of TLR4 signalling parameters did not differ between control and p-EHC-treated horses. Similarly, the p-GI did not result in up-regulation of lamellar protein expression of any parameter. The results suggest that insulin-sensitive tissues may not accurately reflect lamellar pathology during hyperinsulinaemia. While TLR4 is present in the lamellae, its activation appears unlikely to contribute significantly to the developmental pathogenesis of hyperinsulinaemic laminitis. However, inflammation may have a role to play in the later stages (e.g., repair or remodelling) of the disease.